Vaccine development traditionally has focused on the generation of protective antibodies capable of neutralizing infectious agents. To date, the agents used as vaccines typically include inactivated or attenuated microorganisms (for example, bacteria or viruses), their products (for example, toxins), or purified antigens. With the advent of modern molecular biology and gene cloning methodologies, it has been possible to make purer, and apparently more specific vaccines. Furthermore, knowledge of the immune system at a molecular level has permitted the isolation and characterization of immune responses stimulated by infectious agents. Two components of the immune system believed to be central to the successful generation of immune responses include: the pivotal roles of regulatory and cytotoxic T cells; and the manner by which an antigen is presented to these cells by an antigen presenting cell (APC). See, for example, W. E. Paul, ed. (1993) FUNDAMENTALS OF IMMUNOLOGY, Raven Press, Ltd., New York.
Typically, a protein or peptide antigen received from the outside of an APC (exogenous antigen) is degraded within an endocytic vesicle or endosome of the APC, whereupon the resulting peptide fragments form a complex with major histocompatability class (MHC) class II proteins. The resulting complex moves to the cell surface where it is displayed to immune cells neighboring the APC. The peptide fragment fits into a groove defined by the MHC molecule, and the complex may be recognized by a T cell expressing a T cell receptor having binding specificity for the complex. Interaction between a peptide-loaded MHC class II molecule and a helper T cell, referred to in the art as a CD4 T cell, is further stabilized by another interaction between the MHC class II molecule itself and a CD4+ receptor on the surface of the T cell. Thus, exogenous antigen which is processed within APC cells is presented on the cell surface via a MHC class II molecule. The MHC class II complex, when presented to CD4+ T cells, results in the CD4+ helper cell secreting cytokines that stimulate B cells to produce antibodies against the peptide. See, Paul, supra.
Vaccination with exogenous antigen typically results in a CD4 cell-mediated T cell response that generally results in antibody production. Cytotoxic T cells (CTL) typically are not stimulated by such a pathway. Apparently, CTL are stimulated in situations where the antigen originates from inside the APC itself (endogenous antigen), for example, via production of viral proteins in a virally infected cell or cancer-specific proteins in a cancer cell. In fact, in many viral diseases, the generation of CTL is believed to be critical in eliminating virus-infected cells, and thus recovery from infection.
Studies indicate that endogenous and exogenous antigens are processed differently. During synthesis of nascent polypeptides, a portion of the polypeptide is degraded by an intracellular structure called a proteosome. Fragments from this process complex with newly synthesized MHC class I rather than MHC class II molecules, whereupon the resulting antigen containing MHC Class I complexes are transported to the cell surface. Again, T cells with specificity for the specific peptide fragment bind T cells, but in this case, the required co-receptor interaction occurs between MHC class I molecule and a CD8 molecule. Accordingly, endogenous antigen on the surface of the APC is presented to CD8+ T cells. Although there are some types of CD8+ T cells that are not cytotoxic, the CD8+ T cells make up the majority of CTL.
Accordingly, it appears that the design of a vaccine capable of inducing strong CTL responses, requires that the antigenic molecule (generally a protein), either be made inside the cell or delivered into the appropriate cellular compartment so that it can enter the MHC class I processing pathway. One strategy is to incorporate a gene encoding a protein or peptide of interest into a virus, and then use the engineered virus as a vaccine (Lorenz et al. (1999) HUM. GENE THER. 10:623-631). Another strategy is to inject a protein-encoding DNA vector into a cell, and then to administer the cell to the animal or patient where it is expressed from within the cell, and is then presented on the cell surface via MHC class I molecules (Donnelly et al. (1997) ANNU. REV. IMMUNOL. 15:617). A simpler technique of injecting DNA vectors directly into muscle or skin has been shown to induce CTL and/or antibody responses to several antigens (Lai et al. (1988) CRIT. REV. IMMUNOL. 18:449-84 and U.S. Pat. No. 5,589,466). Studies have shown that the antigen is taken up and processed by APC, where it is presented to the immune system (Lai et al., supra).
The delivery of exogenous peptides or proteins to the MHC class I pathway has been partially successful through use of chemical adjuvants such as Freund's adjuvant, and mixtures of squalene and detergents (Hilgers et al. (1999) VACCINE 17:219-228), and more recently through use of small antigen-coated beads which are phagocytosed by macrophages and induce CTL responses via an alternative MHC class I pathway (De Bruijn et al. (1995) EUR. J. IMMUNOL. 25:1274-1285). Furthermore, other methods for enhancing immune responses to an antigen may include the use of chemical adjuvants in combination with recombinant immunostimulatory cytokines, for example, IL-2, IL-12, GM-CSF, and others. For example, one method employs an anti-hapten antibody fused to IL-2 as a way of linking this cytokine to a protein antigen which has been chemically reacted with the hapten (Harvill et al. (1996) J. IMMUNOL. 157:3165).
Another technique exploits antibody “antigenization” whereby a portion of an immunoglobulin variable region is replaced with a peptide antigen. The peptide antigen of the hybrid molecule is presented to an APC once the recombinant antibody binds the APC via interaction with Fc receptors on the surface of the APC (Lanza et al. (1993) PROC. NATL. ACAD. SCI. USA 90:11683-11687). An extension of this approach utilizes splenic injection of plasmid DNA encoding an “antigenized” immunoglobulin heavy chain, after which spleen-derived B cells secrete the recombinant antibody once an immunoglobulin light chain partner is provided.
Immunogenicity of the antigen delivery system, however, is one of the major technical hurdles in modem vaccine development. The goal of vaccination is to elicit a strong immune response. However, because the host immune system has evolved to fight bacteria and viruses, when bacteria or viruses are used as vectors, the messenger typically is destroyed along with the message. Furthermore, strong immune responses to certain viral vectors, for example, vaccinia and adenovirus, limit their utility, and it is contemplated that similar problems can arise during use of bacterial toxins as protein vectors. Likewise, antibody-based “protein vectors” utilizing variable regions that, by their very nature, are not considered as “self” by the immune system are potentially immunogenic. It is contemplated that multiple uses of these carrier molecules can induce anti-idiotypic responses thereby precluding their efficacious use. Accordingly, it is an object of the present invention to provide a vaccine which produces a strong and long lasting immunity against a preselected protein or peptide antigen.